Teratogenic Effects of 5-Chlorodeoxyuridine
on the Rat
Fetus; Protection by Physiological Pyrimidines*
SHAKUNTALA
CHAUBE
(Embryology Section, Division of Experimental
AND M.
Lois
Chemotherapy, Sloan-Kettering
New York)
MURPHY
Institute for Cancer Research, New York,
SUMMARY
Pregnant
Wistar
rats received
single intraperitoneal
dine (C1UdR) on the 12th day of gestation.
injections
of 5-chlorodeoxyuri
They were sacrificed on the 21st day and
the fetuses examined for gross malformations.
For study of skeletal malformations,
specimens were fixed in 95 per cent ethanol, cleared and stained in alizarin red, and
examined in 100 per cent glycerine.
Single doses of 125, 250, 500, and 1000 mg/kg of
C1UdR, which did not cause an excessive number of fetal resorptions, produced mal
formations such as clubbed appendages, poly- and ectrodactylous fore and rear paws,
encephaly, cleft palate, and retarded kinky
survivors at each of these doses, respectively.
tail in 27, 78, 91, and 100 per cent of the
In experiments in which pregnant rats received 500 mg/kg of C1UdR and varying
amounts of TdR simultaneously, doses of TdR below 125 mg/kg did not protect against
the teratogenic
action of C1UdR; but at 250 mg/kg or more of TdR complete protec
tion occurred.
In other experiments
equal amounts (500 mg/kg)
of the two corn
pounds were given separately at time intervals ranging from 15 to 240 minutes.
In
the group of animals
which received
the C1UdR after TdR,
complete
curred up to 30 minutes and partial protection thereafter.
were confined
exclusively
to the rear feet.
When
protection
oc
Resulting abnormalities
C1UdR was given prior to TdR,
only
50 per cent of the surviving embryos were normal at 15 minutes (partial protection)
and none at 90 minutes.
It is adduced that decline in protective activity of TdR is presumably proportional
to the rate of incorporation
its rapid degradation
Recent
investigations
in
a
of C1UdR into the replicating
in embryonic
variety
of
experimental
systems indicate that the type of halogen, fluorine, bro
mine, iodine, or chlorine, substituted
at the 5-position of
uracil deoxyriboside greatly influences the activity and
the type of inhibition of cell proliferation. 5-Fluorodeoxy
uridine (FUdR) inhibits deoxyribonucleic
acid (DNA)
synthesis by blocking the methylation
of deoxyuridylic
acid to form thymidylic acid (10, 19, 20), whereas bromo
deoxyuridine (BUdR), iododeoxyuridine (IUdR), and
chlorodeoxyuridine (C1UdR) inhibit the incorporation of
thymidine (TdR) into DNA (12, 15, 16, 20, 38, 39, 42).
In addition, 5-bromouracil (BU), -iodouracil (113), and
chlorouracil (CR3) substitute for the thymine of DNA
(14, 19, 35, 43, 44), whereas 5-fluorouracil (FU) and
chlorouracil (C1U) are incorporated in place of uradil into
bacterial ribonucleic acid (RNA) (9, 13, 19, 21, 44). The
use of radioactive
forms of the abnormal
@
Research
American
Cancer
Lasker Foundation,
was
supported
in
part
by
pyrimidines
grants
bromo-,
from
Received for publication July 2, 1964.
derivatives
of deoxyuridine
(IUdR,
BUdR),
and
5-fluorodeoxycytidine were shown to be teratogenic in
rat, chick (11, 28), and mouse embryos.1 Thymidine will
prevent the toxicity of FUdR in several in vitro and
embryonic
systems
(19, 27, 29, 34, 40).
In intact
mice,
however, TdR enhanced the toxicity of FUdR (7) whereas
timed administration slightly decreased its teratogenic
effect on the mouse embryo.2 This may be explained by
on
Lois
Murphy,
et
al.,
Teratogenic
Effects
of
Fluorodeoxy
uridine and Fluorodeoxycytidinein Inbred Strains of Mice. Ter
the
Society,
Inc. (T-40), the Albert
and Mary
and grants from the National
Institutes
of
Health (CA03192-08CY).
in place of TdR and
mammalian cell lines in tissue culture (15, 18, 31) in vitro
on E. coli (2, 4, 10) in mouse leukemia (34) and in rats and
mice in vivo (32) indicate that BUdR, IUdR, and C1UdR
may be incorporated in place of TdR in DNA replication;
hence, their inhibitory effects may be partially or corn
pletely reversed in the presence of exogenous TdR in
these systems (2, 4, 10, 15, 18, 31, 34, 40).
The 5-fluoro analogs of uracil, orotic acid, the iodo-,
1 M.
* This
DNA
tissue.
atology
2
Society,
p@ Dagg,
1st Annual
and E. Kallio,
Meeting,
Effects
1961, Abstract
of Thymidine
*7.
on the Tera
togenic and Embryocidal Activity of Fluorodeoxyuridine. Tera
tology Society, 2nd Annual Meeting, 1962,Abstract %1.
1986
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1964 American Association for Cancer Research.
CHATJBE
AND
MURPHY—Teratogenic
TdR's inhibiting the degradation of FUdR in vivo and
thus prolonging its action.
In this study the teratogenic activity of C1UdR in the
developing rat fetus is demonstrated, and TdR is shown
to protect the 12th-day fetus against fetal malformations
produced by 500 mg/kg of C1UdR.
MATERIALS
AND
estrus
by exposure
to males
of the
same
strain.
Pregnant females were caged separately and fed the
standard laboratory chow pellet with water ad libitum.
On the 12th day of gestation the rats were given intraperi
toneal injections of varying single doses (on a mg/kg of
body weight basis) of C1UdR or TdR alone or in combina
tion.
When
the
two
compounds
were
combined,
TABLE 1
C1UdR injected intraperitoneally
rat
c@ wrruspac@nc
injected (mg/kg):
Totalno.examined:62
32123 372503350068700271000
and ectro-@ rear
paw024729797100Retarded
dactylous
f
rear leg
and
clubbed
fore leg0
91Retarded,
027 051 072 2385 44100
tail0018568988Ectro
kinky
500
and syn-'l@fore
paw0001644100Cleft
dactylous
J
interaction
palate000008.5Encephaly0000063
was also investigated.
into 12th-day pregnant
FaTALEriacrPaa
ABNORMALITIESC1Ud.R
mg/kg of C1UdR was injected simultaneously with doses
of TdR ranging in amounts from 7 to 2000 mg/kg. Timed
of the two compounds
1987
of 5-Chlorodeoxyuridine
FETAL MALFORMATIONS OBSERVED ON 21ST DAY OF GESTATION
35Poly-
METHODS
One hundred and thirty-eight female rats of the CF
Wistar strain, weighing from 170 to 250 gm., were mated
during
Effects
In these experiments 500 mg/kg of C1UdR was injected at
times varying from 0 to 240 minutes before or after a single
dose of 250 or 500 mg/kg of TdR. The animals were
sacrificed
@
on the 21st day of gestation;
surviving
fetuses
normal survivors was recovered at 21 days from litters
were removed from the uteri, weighed and examined for
whose mothers
malformations,
12th day.
and approximately
half the number
from
were treated
with 125-1000
mg/kg
on the
The types of abnormalities frequently seen at
each litter were fixed in 95 per cent ethanol, cleared, and
subsequently stained in alizarin red. Stained specimens
were examined in 100 per cent glycerine for the presence
these doses are listed in Table 1 ; there is an increasing
incidence of specific abnormalities
and a more extensive
of skeletal anomalies.
The number of dead and resorbed
fetuses in each litter was also recorded.
C1UdR and TdR
were dissolved in distilled water and used within
hour
doses of the drug.
after preparation.
Representative embryos from litters treated with 500
and 1000 mg/kg of C1UdR are shown in Figures 1 and 2
and associated skeletal deformities in Figures 3 and 4.
By comparison, 250 mg/kg of C1UdR produced no
teratogenic effect on the 11th day, and 500 mg/kg gave
only 35 per cent abnormal survivors, which showed only
minor tail defects and polydactyly of the rear paws. At
RESULTS
The per cent of fetal mortality observed and the per
cent of abnormal survivors that were obtained at different
doses of C1UdR are shown in Chart 1. Fetal mortality
involvement of different parts of the body with higher
At 125 and 250 mg/kg,
were predominantly
abnormalities
of the rear appendages, whereas at
500 and 700 mg/kg
the entire
embryo
was deformed.
for all the doses of C1UdR shown in the graph (62—1000
mg/kg) were within the normal (n) control range of 0—10 1000 mg/kg, 95 per cent of the fetuses at 21 days were
abnormal, with additional cleft palate and lip.
per cent.
A dose of 62 mg/kg did not produce any mal
formations, but an increasingly higher percentage of ab
pregnant females
very small.
100
@60
E
0
@40
TdR
C
5,
I.,
@@20
mortality
0
Minutes
TdR before
CIUdR
CHART 1.—Lethal and teratogenic
intrapertioneal
tolerated
1000 mg/kg
but fetuses
were
Thymidine alone, at single doses of 500, 1000, or 2000
mg/kg, injected into the 12th-day pregnant rat did not
cause fetal mortality or malformations.
C1UdR and TdR were given in various time sequences.
The results of experiments in which 500 mg/kg of C1UdR
was injected simultaneously with varying amounts of
0
>
>
C
While selecting doses for the study, it was noted that
40-gm. rats showed no ifi effects from 2000 mg/kg and
injections
of C1UdR
rat, sacrificedon the 21st day.
TcIR
after
CIUdR
effects produced
by single
on the
pregnant
12th
day
on the
12th
day
of gestation
are summarized
in
Table 2. From these data it is evident that a minimal
dose of 250 mg/kg of TdR is required to provide complete
protection of the fetus against 500 mg/kg of C1TJdR, but
lower
doses
(7—125 mg/kg)
also appear
to be partially
effective in reducing the number of fetuses with all ab
normalities listed in Table 2 except that of the rear paw.
This is particularly evident when 7 mg/kg of thymidine
was given.
Fetal
mortality
in this experimental
series
was within the 0—10
per cent normal range.
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1964 American Association for Cancer Research.
Cancer Research
1988
Vol. 24, December
1964
TABLE 2
PROTECTIVE
EFFECTS
OF VARYING
AMOUNTS
OF TDR
AGAINST
500 MG/KG
OF CLUDR
WHEN
INJECTED
SIMULTANEOUSLY INTO 12-DAY PREGNANT RAT
FETAL
EFFECTSPER
ABNORMALITIESTdR
CENTWITHSPECIFIC
(mg/kg)
Total survivors:
Per cent abnormal:0
68
16
33
35
45
35
30
36
917 10015 10031 10062 100125 92250 0500 0
paw979494100100970Retarded
0Poly- and ectrodactylous:rear
0Retarded,
15
0
36
0
33
0
0700 0100002000
and clubbed@°―@leg
Jforeleg72
2362 1822 1113
611
50
00
00
00
00
00
tail251132300000Ectrokinky
and syndactylous:fore
paw1612800000000
The mortality was between 0 and 10 per cent in all groups (control value).
partial protection
(50 per cent
cent at 240 minutes)
thereafter;
at 90 minutes
and 25 per
with 250 mg/kg of TdR
64 per cent of the survivors were normal at 15 minutes
and none at 90 minutes. When 500 mg/kg of TdR was
given
up to 30 minutes
the survivors
survivors
80
after
C1UdR,
only 50 per cent of
were normal and none at 90 minutes.
Ab
normalities in all partially protected fetuses were con
fined exclusively to the rear appendages (Table 3).
DISCUSSION
The data presented demonstrate that the rat fetus can be
60
severely injured by brief periods of exposure
of C1UdR.
The proportion
of abnormal
to the action
embryos
ob
tained with single injections of C1UdR given to pregnant
C
4@
rats on the 12th day varied
a,
0.
the widest range of fetal abnormalities occurred with
500 and 1000 mg/kg. With 500 mg/kg consistent ab
40
normalities
of the
with the dose of the drug,
appendages
and
tail
occurred
and
in over
90 per cent of the survivors at 21 days, whereas with 62.5
mg/kg all treated litters were normal. A dose of 1000
mg/kg of TdR did not produce any fetal malformations.
The protective role of exogenous TdR against halo
genated pyrimidines is well established in several systems
(2, 4, 6, 10, 12, 18, 31, 34, 40). Our experiments show
that 250 mg/kg of TdR given at zero time protects the
20
12th-day
fetus
against
the
teratogenic
effects
of 500
mg/kg of C1UdR, thus indicating that this amount of
TdR
1000
CHART 2.—Fetal
effects
produced
by timed
counteracts
at least
400 mg/kg
of C1UdR.
This
effectiveness of TdR to protect the embryo against C1UdR,
CIUdRmg/kg
interaction
of a
single dose of 500 mg/kg of C1UdR and 250 or 500 mg/kg of TdR
in the 12th-day pregnant rat. Sacrificed on the 21st day.
Further evidence in support of the protective role of
however,
declines
gradually
between administration
with increasing
time intervals
of the two compounds.
Thus at
15 minutes 500 mg/kg of TdR (in experiments
in which
TdR is given after C1UdR) gives only partial protection
in which a
(50 per cent abnormal survivors), which is equivalent to
the effect seen with 125 mg/kg of C1UdR alone; this sug
single dose of 250 or 500 mg/kg of TdR was injected
into rats prior to or after an injection of 500 mg/kg of
C1UdR at time intervals of 15, 30, 60, 90, 150, 210, or
been completed. In the same experimental series 500
mg/kg of TdR at 60 minutes after C1UdR gives about 95
240 minutes.
per cent abnormal
TdR was obtained
from a series of experiments
The results
are represented
graphically
in
Chart 2. A dose of 500 mg/kg of TdR, given prior to
C1UdR, provided a complete protection against the
teratogenic
effect of the latter up to 30 minutes
and only
gests that about 25 per cent of C1UdR effect has already
effect seen between
fetuses,
and this is equivalent
250 and 500 mg/kg
to the
(78—92per cent
abnormal fetuses) of C1UdR alone. This implies that
the teratogenic effect of C1UdR is almost completed by
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1964 American Association for Cancer Research.
CHAUBE
AND
MutPHY—Teratogenic
Effects
1989
of 5-Chlorodeoxyuridine
TABLE 3
PROTECTIVE
EFFECTS
OF TDR
WHEN
GIvEN
AT VARIOUS
TIMES
BEFORE
AND
AFTER
500 mg/kg
of
CLUDR @roTHE 12-DAY PREGNANTRAT
MINUTES
PRIOR
AFRERCIUdR
(500
15-3060-90TdR
90-240
60
30
15
(500
@@@g@kg)TdR
mgJkg)—
0MINUTES
(500 mg/kg):
Per cent abnormal
survivors
Per cent appendicular malformations:
Poly- and ectrodactylous
(rear paw)
100
100
100
0
0
0
00
0
050
100
97
Retarded and/or clubbed:
Rear leg
230TdRForeleg54—75
0
014
0
00
0
3—10
095—100
091
72
(250 mg/kg):
Per cent abnormal survivors
Per cent appendicular
malformations:
Poly- and ectodactylous (rear paw)
Retarded
60 minutes and, once completed, is irreversible.
of decline of protective
proportional
100
and clubbed (rear leg)100
activity
to the rate
The rate
significant
amounts
of C1UdR are incorporated into the replicating DNA of the
embryonic
cells in place of TdR.
A further
suggestion
of the rate of catabolism of C1UdR may be adduced from
the data on the decreasing protective effect with time
following C1UdR. Despite the fact that C1UdR is avail
able for only a short time, its damaging effect on the
fetus is irreversible.
When TdR is given 60 or more
minutes before C1UdR there is a rapid loss of protective
activity
against
C1UdR.
This observation
is consistent
with a report of the availability of exogenous thymidine.
04-labeled thymidine was cleared rapidly from blood of
rats ; only 2 per cent of an intravenous dose remained at
1 hour (32).
whereas this
TdR did not potentiate the effect of C1UdR,
effect occurs with FUdR (7) ; this supports
the impression that FUdR and C1UdR act by different
mechanisms.
Tracer
experiments
have
demonstrated
that
DNA
is
biochemically inactive in tissues where little or no cell
division
is occurring.
However,
in proliferating
tissues,
such as tumors (1) and regenerating liver (22—24,26, 36),
labeled precursors are rapidly incorporated into the DNA.
In vitro dividing
cells utilize
nucleosides
added
to the
medium for nucleic acid synthesis (41). It has been sug
gested that thymidine kinase may be playing an important
role in controlling
DNA
synthesis
in regenerating
a*s well as in tissues of fetal and neonatal
liver,
rats, by con
trolling the synthesis of dTTP (3, 8, 25, 33, 37). Further
more, stimulation of mitotic activity (17) and elevation
of thymidine
kinase specificity
(25) were observed
normal
rat tissues
following
TdR
injection.
The
in
rat
embryo as a rapidly proliferating tissue appears to be
more
sensitive
sumably
Reports
anti-cancer
to C1UdR
than
the
by its relative requirements
concerning
drugs
cellular
suggest
that
pregnant
rat,
pre
for TdR and kinase.
inhibition
produced
cells in general
362
by
fail to
00 00
distinguish between the natural metabolite and certain
analogs.
of TdR is thus presumably
at which
100 100 0
15.683
C1UdR acted as an effective antimetabolite
in
the embryonic system under study and was presumably
incorporated into the DNA of the cells in place of TdR
and also acted to inhibit pyrimidine utilization. Em
bryonic damage is probably
due to alteration
of DNA by
C1UdR incorporation, since a delay in TdR incorporation
into DNA would not be expected to result in such severe
injury,
irreversible
within
60 minutes
by TCIR.
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and Related
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samples
of fetuses
A , control,
and B
and C, from litters treated with 500 and 1000 mg/kg of C1UdR,
respectively, on the 12th day and sacrificed on the 21st day of
gestation.
In addition to clubbed rear appendages, polydactylous
rear paws,
ectrodactylous
fore paws,
and retarded
kinky
tail in
both B and C, example C had exencephaly and cleft palate.
Fia. 2.—Rearand fore paws of fetuses from a control, a and d
litter and from litters treated with 500 mg/kg of C1UdR, b, c, and
e, f on the 12th day and sacrificed on the 21st day; b, note the
preaxial
hyperphalangy
(macro + poly —dactyly)
; c, ectrodactyly
with absence of the 4th and 5th digits ; e, ectrodactyly
1st and 5th digits
were absent;
in which
f, adactyly.
Fia. 3.—Dorsal view of the fetal skeleton stained in alizarin
red.
A , control;
B and C from litters
treated
with 500 and 1000
mg/kg of C1UdR, respectively, on the 12th day and sacrificed on
the 21st day of gestation.
Vertebral centra, sternebrae,
and costal
portions of the ribs were removed for the purpose of photography.
Beside general retardation of the entire skeleton, shortening of the
principal
bones
of the
appendages
and pelvis,
and incomplete
ossification of several bones of the skull which occurred at both
doses,
specimen
C had fused ribs, absence
of the bony palate,
in
dented scapula (rt. side), and partially ossified cervical vertebrae
and sternebrae.
FIG. 4.—Skeleton of the fore and rear legs stained in alizarin
red.
a and d, controls; b, c and d from litters treated with 500
mg/kg of C1UdR. Note the absence of the deltoid ridge of the
humerus
and
absent
metacarpals
in b and c and
retardation
of
tibia and fibula and presence of extra metatarsal associated with
duplication of the 1st toe in e.
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1964 American Association for Cancer Research.
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Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1964 American Association for Cancer Research.
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Teratogenic Effects of 5-Chlorodeoxyuridine on the Rat Fetus:
Protection by Physiological Pyrimidines
Shakuntala Chaube and M. Lois Murphy
Cancer Res 1964;24:1986-1993.
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